Exposure to chronic hypoxia (CH) occurs with many pulmonary diseases and results in the development of pulmonary hypertension. Studies from our lab and others demonstrated a key role for the transcription factor, hypoxia-inducible factor-1 (HIF-1) in the development of hypoxic pulmonary hypertension. It is well recognized that expression of HIF-1a, the oxygen-sensitive subunit of HIF-1, correlates with hypoxic induction of genes encoding factors implicated in development of pulmonary hypertension, including endothelin-1 (ET-1), a potent vasoconstrictive and mitogenic agent. During the previous funding period, we defined several mechanisms by which CH and ET-1 alter pulmonary vasomotor tone. Recently, our studies revealed a new paradigm where ET-1 regulates HIF-1 expression. Our preliminary data show that ET-1 increased expression of the oxygen-sensitive 1 subunit of HIF-1, HIF-1a, in pulmonary arterial smooth muscle cells (PASMCs), even under normoxic conditions, and reduced expression of prolyl hydroxylases, key enzymes that are responsible for targeting HIF-1a for rapid degradation. These data suggest that while activation of HIF-1 by hypoxia in endothelial cells might cause elevated ET-1 production, subsequent ET-1 signaling in PASMCs contributes to maintained upregulation of HIF-1, creating a positive feedback, or feed-forward, process. Conversely, an elevation in ET-1 levels, as occurs in numerous disease states, may result in increased HIF-1 expression in the absence of associated hypoxia. Based on these new findings, we hypothesize that during moderate hypoxia, increased pulmonary ET-1 production and activation of ET-1 receptors on PASMCs leads to a positive feedback, or feed- forward, mechanism of HIF-11 protein accumulation and enhanced HIF-1-dependent gene transcription. This results in alterations in PASMC function which contribute to the development of pulmonary hypertension. To test this hypothesis, we will use a combination of techniques including transgenic animals, microfluorescence measurements, whole-cell patch-clamp, and molecular biology, to accomplish the following Specific Aims: 1) determine whether ET-1 derived specifically from endothelial cells is required for and/or accelerates HIF-dependent pathophysiological effects of CH in the pulmonary circulation;2) elucidate the mechanism(s) by which ET-1 modulates HIF-1 expression and 3) determine whether HIF-1 is the downstream effector molecule mediating hypoxia- induced alterations in PASMC homeostasis. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore cellular mechanisms involved in the development of pulmonary hypertension, a devastating disease with limited treatment options. Understanding the cellular changes that occur in the pulmonary vasculature with development of pulmonary hypertension is key to advancing treatment and therapeutic options.